PCR Woes.

[Roland Hubner]

> I am trying to PCR up a 3.5kb insert from a plasmid and its not working.
> Are there any fundamental changes to parameters I need to implement in
> order for it to work. I am using pfu with extension times of up to 8
> minutes and longer. I have tried it at 52, 54, and 58 degrees. I also
> decreased my extension temp to 68 to see if that would work, but no.
> 
> Can you help?
> Best wishes
> John              

John,

 you need to give more info about your exptl. conditions...

 Does it work in your hands with Taq or Tfl and 4 min extensions?

Greetings,
 Roland